The present invention is directed to compositions comprising a peptide nucleic acid (PNA) which is conjugated to a lipophilic group and incorporated into liposomes. The PNA is composed of naturally-occurring nucleobases or non-naturally-occurring nucleobases which are covalently bound to a polyamide backbone. The PNA compositions of the present invention may further comprise a PNA modified by an amino acid side chain. The PNA compositions of the present invention exhibit enhanced cellular uptake and distribution. PNA compositions which were incorporated into liposomes demonstrated increased cellular uptake and more diffuse distribution than PNA compositions without liposomes.
The function of a gene starts by transcription of its information to a messenger RNA (mRNA). By interacting with the ribosomal complex, mRNA directs synthesis of proteins. This protein synthesis process is known as translation. Translation requires the presence of various cofactors, building blocks, amino acids and transfer RNAs (tRNAs), all of which are present in normal cells.
Most conventional drugs exert their effect by interacting with and modulating one or more targeted endogenous proteins, e.g., enzymes. Typically, however, such drugs are not specific for targeted proteins but interact with other proteins as well. Thus, use of a relatively large dose of drug is necessary to effectively modulate the action of a particular protein. If the modulation of a protein activity could be achieved by interaction with or inactivation of mRNA, a dramatic reduction in the amount of drug necessary and in the side-effects of the drug could be achieved. Further reductions in the amount of drug necessary and the side-effects could be obtained if such interaction is site-specific. Since a functioning gene continually produces mRNA, it would be even more advantageous if gene transcription could be arrested in its entirety. Oligonucleotides and their analogs have been developed and used as diagnostics, therapeutics and research reagents. One example of a modification to oligonucleotides is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and 2xe2x80x2-O-methyl ribose sugar moieties. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. Phosphorothioate oligonucleotides are presently being used as antisense agents in human clinical trials for the treatment of various disease states. Although some improvements in diagnostic and therapeutic uses have been realized with these oligonucleotide modifications, there exists an ongoing demand for improved oligonucleotide analogs.
There are several known nucleic acid analogs having nucleobases bound to backbones other than the naturally-occurring ribonucleic acids or deoxyribonucleic acids. These nucleic acid analogs have the ability to bind to nucleic acids with complementary nucleobase sequences. Among these, the peptide nucleic acids (PNAs), as described, for example, in WO 92/20702, have been shown to be useful as therapeutic and diagnostic reagents. This may be due to their generally higher affinity for complementary nucleobase sequence than the corresponding wild-type nucleic acids.
PNAs are useful surrogates for oligonucleotides in binding to DNA and RNA. Egholm et al., Nature, 1993, 365, 566, and references cited therein. The current literature reflects the various applications of PNAs. Hyrup et al., Bioorganic and Med. Chem., 1996, 4, 5; and Nielsen, Perspectives Drug Disc. Des., 1996, 4, 76.
PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced by a peptide backbone. Each subunit of the peptide backbone is attached to a naturally-occurring or non-naturally-occurring nucleobase. One such peptide backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. The synthesis of PNAs via preformed monomers was previously described in WO 92/20702 and WO 92/20703, the contents of which are herein incorporated by reference. More recent advances in the structure and synthesis of PNAs are illustrated in WO 93/12129 and U.S. Pat. No. 5,539,082, issued Jul. 23, 1996, the contents of both being herein incorporated by reference. Further, the literature is replete with publications describing synthetic procedures, biological properties and uses of PNAs. For example, PNAs possess the ability to effect strand displacement of double-stranded DNA. Patel. Nature, 1993, 365, 490. Improved synthetic procedures for PNAs have also been described. Nielsen et al., Science, 1991, 254, 1497; and Egholm, J. Am. Chem. Soc., 1992, 114, 1895. PNAs form duplexes and triplexes with complementary DNA or RNA. Knudson et al., Nucleic Acids Research, 1996, 24, 494; Nielsen et al., J. Am. Chem. Soc., 1996, 118, 2287; Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; and Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677.
PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes. The resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (Tm). This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA) duplexes is that Tm is practically independent of salt concentration. DNA/DNA duplexes, on the other hand, are highly dependent on the ionic strength.
Triplex formation by oligonucleotides has been an area of intense investigation since sequence-specific cleavage of double-stranded deoxyribonucleic acid (DNA) was demonstrated. Moser et al., Science, 1987, 238, 645. The potential use of triplex-forming oligonucleotides in gene therapy, diagnostic probing, and other biomedical applications has generated considerable interest Uhlmann et al., Chemical Reviews, 1990, 90, 543. Pyrimidine oligonucleotides have been shown to form triple helix structures through binding to homopurine targets in double-stranded DNA. In these structures the new pyrimidine strand is oriented parallel to the purine Watson-Crick strand in the major groove of the DNA and binds through sequence-specific Hoogsteen hydrogen bonding. The sequence specificity is derived from thymine recognizing adenine (T:A-T) and protonated cytosine recognizing guanine (C+:G-C). Best et al., J. Am. Chem. Soc., 1995, 117, 1187. In a less well-studied triplex motif, purine-rich oligonucleotides bind to purine targets of double-stranded DNA. The orientation of the third strand in this motif is anti-parallel to the purine Watson-Crick strand, and the specificity is derived from guanine recognizing guanine (G:G-C) and thymine or adenine recognizing adenine (A:A-T or T:A-T). Greenberg et al., J. Am. Chem. Soc., 1995, 117, 5016.
Homopyrimidine PNAs have been shown to bind complementary DNA or RNA forming (PNA)2/DNA(RNA) triplexes of high thermal stability. Egholm et al., Science, 1991, 254, 1497; Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm et al., J. Am. Chem. Soc., 1992, 114, 9677. The formation of triplexes involving two PNA strands and one nucleotide strand has been reported in U.S. patent application Ser. No. 08/088,661, filed Jul. 2, 1993, the contents of which are incorporated herein by reference. The formation of triplexes in which the Hoogsteen strand is parallel to the DNA purine target strand is preferred to formation of anti-parallel complexes. This allows for the use of bis-PNAs to obtain triple helix structures with increased pH-independent thermal stability using pseudoisocytosine instead of cytosine in the Hoogsteen strand. Egholm et al., J. Am. Chem. Soc., 1992, 114, 1895. Further, see WO 96/02558, the contents of which are incorporated herein by reference.
Peptide nucleic acids have been shown to have higher binding affinities (as determined by their Tm""s) for both DNA and RNA than that of DNA or RNA to either DNA or RNA. This increase in binding affinity makes these peptide nucleic acid oligomers especially useful as molecular probes and diagnostic agents for nucleic acid species.
In addition to increased affinity, PNAs have increased specificity for DNA binding. Thus, a PNA/DNA duplex mismatch show 8 to 20xc2x0 C. drop in the Tm relative to the DNA/DNA duplex. This decrease in Tm is not observed with the corresponding DNA/DNA duplex mismatch. Egholm et al., Nature 1993, 365, 566.
A further advantage of PNAs, compared to oligonucleotides, is that the polyamide backbone of PNAs is resistant to degradation by enzymes.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs that bind to complementary DNA and RNA strands for use as diagnostics, research reagents and potential therapeutics. For many applications, the oligonucleotides and oligonucleotide analogs must be transported across cell membranes or taken up by cells to express their activity.
PCT/EP/01219 describes novel PNAs which bind to complementary DNA and RNA more tightly than the corresponding DNA. It is desirable to append groups to these PNAs which will modulate their activity, modify their membrane permeability or increase their cellular uptake property. One method for increasing amount of cellular uptake property of PNAs is to attach a lipophilic group. U.S. application Ser. No. 117,363, filed Sep. 3, 1993, describes several alkylamino functionalities and their use in the attachment of such pendant groups to oligonucleosides.
U.S. application Ser. No. 07/943,516, filed Sep. 11, 1992, and its corresponding published PCT application WO 94/06815, describe other novel amine-containing compounds and their incorporation into oligonucleotides for, inter alia, the purposes of enhancing cellular uptake, increasing lipophilicity, causing greater cellular retention and increasing the distribution of the compound within the cell.
U.S. application Ser. No. 08/116,801, filed Sep. 3, 1993, describes nucleosides and oligonucleosides derivatized to include a thiolalkyl functionality, through which pendant groups are attached.
Recently, liposomal drug-delivery systems incorporating various biomolecules and drugs have been studied and found to exhibit reduced toxicities and increased efficacy due to enhanced cellular uptake and distribution. Chonn and Cullis, Current Opinion in Biotechnology, 1995, 6, 698; Mannino et al., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem et Biophys. Acta, 1990, 1029, 91; and Lappalainen et al., Antiviral Res., 1994, 23, 119. Liposomes are microscopic spheres composed of an aqueous core and a lipid bilayer enveloping the core. Procedures for preparation of liposomes are available in the literature. G. Gregoridadis in xe2x80x9cLiposome Technology,xe2x80x9d volume 2, G. Gregoridadis (ed.), CRC Press, 1993, p. 1; Watwe and Bellare, Curr. Sci., 1995, 68, 715. Several liposomal drugs are currently on the market or under development. Chonn and Cullis, Current Opinion in Biotechnology, 1995, 6, 698.
WO 96/10391, published Apr. 11, 1995, describes polyethylene glycol-modified ceramide lipids which are used to form liposomes, and the use of these liposomes as drug-delivery vehicles.
WO 96/24334, published Aug. 15, 1996, describes lipid constructs having an aminomannose-derivatized cholesterol moiety for the delivery of drugs to the cytoplasm of cells, particularly to vascular smooth muscle tissues.
WO 96/40627, published Dec. 19, 1996, describes cationic lipid-containing liposome formulations which are useful in the delivery of biomolecules such as oligonucleotides, nucleic acids, peptides and other agents.
Despite recent advances, there remains a need for stable compositions with enhanced cellular uptake and distribution.
The present invention provides peptide nucleic acids (PNAs) conjugated to a lipophilic group and having a modified backbone wherein an amino acid side chain is attached to the backbone. The present invention also provides liposomal compositions comprising a peptide nucleic acid (PNA) conjugated to a lipophilic group which is incorporated into liposomes. The PNAs of the present invention comprise nucleobases covalently bound to a polyamide backbone. Representative nucleobases include the four major naturally-occurring DNA nucleobases (i.e., thymine, cytosine, adenine and guanine), other naturally-occurring nucleobases (e.g. inosine, uracil, 5-methylcytosine, thiouracil and 2,6diaminopurine) and artificial nucleobases (e.g., bromothymine, azaadenines and azaguanines). These nucleobases are attached to a polyamide backbone through, a suitable linker.
Preferred peptide nucleic acids of the invention have the general formula (I): 
wherein:
each L is, independently, a naturally-occurring nucleobase or a non-naturally-occurring nucleobase;
each R7xe2x80x2 is hydrogen or the side chain of a naturally-occurring or non-naturally-occurring amino acid, at least one R7xe2x80x2 being the side chain of an amino acid;
Rh is OH, NH2, or NHLysNH2;
each of Ri and Rj is, independently, a lipophilic group or an amino acid labeled with a fluorescent group; or Ri and Rj, together, are a lipophilic group;
n is an integer from 1 to 30.
PNAs having formula (I) wherein Ri is D-lysine labeled with a fluorescent group and Rj is an adamantoyl group are preferred. Even more preferred are PNAs of formula (I) wherein Ri is D-lysine labeled with fluorescein and Rj is an adamantoyl group. Also preferred are PNAs having formula (I) wherein Ri and Rj, together, are an adamantoyl group. Further preferred are PNAs of formula (I) wherein at least one of said R7xe2x80x2 is the side chain of D-lysine.
Preferably, the carbon atom to which substituent R7xe2x80x2 is attached is stereochemically enriched. Hereinafter, xe2x80x9cstereochemically enrichedxe2x80x9d means that one stereoisomer predominates over the other stereoisomer in a sufficient amount as to provide a beneficial effect. Preferably, one stereoisomer predominates by more than 50%. More preferably, one stereoisomer predominates by more than 80%. Even more preferably, one stereoisomer predominates by more than 90%. Still more preferably, one stereoisomer predominates by more than 95%. Even more preferably, one stereoisomer predominates by more than 99%. Still even more preferably, one stereoisomer is present substantially quantitatively.
The present invention also provides liposomal compositions comprising a peptide nucleic acid incorporated in a liposome, said peptide nucleic acid having formula (I) wherein:
each L is, independently, a naturally-occurring nucleobase or a non-naturally-occurring nucleobase;
each R7xe2x80x2 is hydrogen or the side chain of a naturally-occurring or non-naturally-occurring amino acid;
Rh is OH, NH2, or NHLysNH2;
each of Ri and Rj is, independently, a lipophilic group or an amino acid labeled with a fluorescent group; or Ri and Rj, together, are a lipophilic group;
n is an integer from 1 to 30.
PNAs having formula (I) wherein Ri is D-lysine labeled with a fluorescent group and Rj is an adamantoyl group are preferred. Even more preferred are PNAs of formula (I) wherein Ri is D-lysine labeled with fluorescein and Rj is an adamantoyl group. Also preferred are PNAs having formula (I) wherein Ri and Rj, together, are an adamantoyl group. Further preferred are PNAs of formula (I) wherein at least one of said R7xe2x80x2 is the side chain of D-lysine.
Preferably, the carbon atom to which substituent R7xe2x80x2 is attached is stereochemically enriched.
The PNAs of the present invention are synthesized by adaptation of standard peptide synthesis procedures, either in solution or on a solid phase.
The present invention further provides methods for enhancing the cellular uptake and distribution of peptide nucleic acids by incorporation of amino acid side chains into PNA backbones, conjugating lipophilic groups with PNAs and introducing PNAs into liposomes.